The specific aims of this grant are directed towards a somatic cell genetic analysis of the Ku86:KARP-1 locus in human cells. In the mouse, Ku86, along with Ku70, the DNA-dependent protein kinase catalytic subunit (DNA-PK[CS]), XRCC-4, DNA ligase IV and Artemis, are required for a critical form of DNA double strand break (DSB) repair known as nonhomologous end joining (NHEJ). Recently, we have shown that Ku86:KARP-1 plays an additional, essential role in human somatic cells. In this grant application we provide preliminary data to demonstrate that this essential role is probably related to the ability of Ku86:KARP-1 to regulate telomere length and genomic stability. This is the first demonstration that one of the NHEJ factors regulates these two processes in human cells. We describe below experiments that elucidate the genetic and molecular role(s) of Ku86 and KARP-1 in DNA DSB repair, telomere length regulation and genomic stability in human cells. The importance of identifying and understanding the genes that control human DNA repair is underscored by the existence of a large number of cancer predisposition syndromes such as ataxia telangiectasia, ataxia telangiectasia-like disorder, Nijmegen Breakage syndrome, Fanconi's anemia, Li-Fraumeni syndrome, xeroderma pigmentosum and breast and colon cancer where it appears that the underlying molecular defects reside in DNA repair genes. We intend to characterize these important pathways using four lines of experimentation: 1. Construction of conditional human Ku86:KARP-1-null cell lines. 2. Why do human Ku86:KARP-1-null tissue culture human cells undergo apoptosis? 3. Is the loss of DNA-PK activity responsible for the Ku86:KARP-1-null phenotypes? 4. Is telomerase biogenesis or function aberrant in Ku86:KARP-1 mutant cells? The ultimate goal of these studies is to use Ku86 and KARP-1 mutant cell lines as tools to understand the molecular mechanisms of DNA DSB repair in humans.